Elucidation of the catalytic mechanism and reaction chemistry of asparagine synthetase will allow the rational design asparagine synthetase specific inhibitors. Based on the efficacy of L-asparaginase as a chemotherapeutic, limited primarily to the treatment of acute lymphoblastic leukemia, it is proposed that inhibitors of asparagine synthetase could act as antineoplastic agents effective against a wide range of tumors. In order to understand better the asparagine synthetase reaction mechanism, systems have been developed for efficient large scale production and purification of human and E. coli asparagine synthetases. Structural, kinetic, and mutagenic studies have been proposed which will test a series of mechanistic hypotheses. The studies will focus upon the mechanism by which the nitrogen of glutamine is transferred to the activated aspartic acid residue. The experimental design includes studies of possible intermediates, as well as mutagenesis of totally conserved residues postulated to have specific functions in catalysis. Extensive kinetic examination of the asparagine synthetase reaction will be used to eliminate possible chemical mechanisms, as well as to define fully the mechanism by which asparagine synthetase transfers the nitrogen from glutamine to the activated form of aspartic acid. The structure of glutamine when bound to the active site of the asparagine synthetase will then be studied by synthesizing a series of glutamine analogs, and testing them as substrates or inhibitors. In addition, a series of possible enzyme inhibitors, with structures based upon putative transition states or reaction intermediates derived from the results of kinetic experiments and theoretical calculations, will be synthesized as potential inhibitors of the reaction. Together, these studies will provide the basis for rational development of antitumor drugs designed to inhibit asparagine biosynthesis.